In vivo evaluation of carbon fullerene toxicity using embryonic zebrafish

Abstract

There is a pressing need to develop rapid whole animal-based testing assays to assess the potential toxicity of engineered nanomaterials. To meet this challenge, the embryonic zebrafish model was employed to determine the toxicity of fullerenes. Embryonic zebrafish were exposed to graded concentrations of fullerenes [C(60), C(70), and C(60)(OH)(24)] during early embryogenesis and the resulting morphological and cellular responses were defined. Exposure to 200 μg/L C(60) and C(70) induced a significant increased in malformations, pericardial edema, and mortality; while the response to C(60)(OH)(24) exposure was less pronounced at concentrations an order of magnitude higher. Exposure to C(60) induced both necrotic and apoptotic cellular death throughout the embryo. While C(60)(OH)(24) induced an increase in embryonic cellular death, it did not induce apoptosis. Our findings concur with results obtained in other models indicating that C(60)(OH)(24) is significantly less toxic than C(60). These studies also suggest that that the embryonic zebrafish model is well-suited for the rapid assessment of nanomaterial toxicity.

Figure 1

Photon correlation spectroscopy of C₆₀ (black) and C₇₀ (gray) at 50 and 200 ppb, and C₆₀(OH)₂₄ (white) at 250 and 1000 ppb. Mode size of particles (highest number of observations) for each concentration is indicated by (•). Boxes represent particle size distribution with upper and lower standard deviation determined by size distribution processor analysis.

Figure 2

Concentration-responses observed for embryonic zebrafish exposed to (a) C₆₀, (b) C₇₀, and (c) C₆₀(OH)₂₄ from 24 hpf to 96 hpf; evaluated daily until 6 dpf. Values represent the % showing the effect by day 6 (Cumulative % Effect): mortality, pericardial edema, yolk sac edema, and fin malformations. Representative images of the caudal fins for (d) control and (e) 200 ppb C₆₀-exposed animals are given. Representative images of the pectoral fin for (f) control and (g) 3500 ppb C₆₀(OH)₂₄-exposed animals. Representative images of (h) 1% DMSO control head at 6 dpf and (i) 200 ppb C₆₀-exposed head at 6 dpf; arrows designate pericardial edema (PE) and yolk sac edema (YSE). Significance was determined using Fisher’s Exact test (*p<0.05) compared to 1% DMSO control (N=24).

Figure 3. C₆₀-exposure leads to increased cellular death in the live zebrafish embryo

Cellular death was determined using acridine orange staining of C₆₀-exposed embryos at 36 hpf, after a 12 hour C₆₀ exposure. Fluorescence emitted from cells undergoing cellular death indicated by white signal on a black background, shown for the head region for (a) 1% DMSO control, (b) 50 ppb C₆₀-exposed (c) 100 ppb C₆₀-exposed (d) 200 ppb C₆₀-exposed; and the caudal fin for (e) 1% DMSO control, (f) 50 ppb C₆₀-exposed, (g) 100 ppb C₆₀-exposed, and (h) 200 ppb C₆₀-exposed embryos. (i) Concentration-response curves for cell death measured as relative fluorescence in the head (•) and trunk regions (°) of the embryos. Cellular death was significantly different than controls at 100 and 200 ppb determined using one-way ANOVA (*p<0.05), N=12

Figure 4

Apoptotic cell death was measured in embryos at 36 hpf, after 12 hours of exposure. White spots indicating a TUNEL⁺ response (apoptotic signaling) shown for the head region (a) 1% DMSO control, (b) 50 ppb C₆₀-exposed (c) 100 ppb C₆₀-exposed (d) 200 ppb C₆₀-exposed and the caudal fin region (e) 1% DMSO control, (f) 50 ppb C₆₀-exposed, (g) 100 ppb C₆₀-exposed, and (h) 200 ppb C₆₀-exposed embryos. (i) Concentration-response curves for cell death measured as relative fluorescence in the head (black) and trunk (grey) regions of the embryos. Apoptotic cellular death was significantly different than controls at 100 and 200 ppb determined using one-way ANOVA (*p<0.05), N=12.